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Identifying phosphorylation sites in culprit proteins and figuring out if they matter for disease progression are no small feats. The situation gets murkier still when certain sites adopt cis or trans conformations, rendering the phosphoprotein unrecognizable to kinases directed at the opposing isomer. Prolyl isomerases help proteins shift from one conformation to another, and one such enzyme—Pin1—seems important in Alzheimer’s disease because it binds phospho-tau and relieves tau pathology in mice. To examine tau isomerization in vivo, researchers led by Kun Ping Lu and Xiao Zhen Zhou at Beth Israel Deaconess Medical Center, Boston, generated antibodies specific for cis and trans forms of phosphorylated tau. In yesterday’s Cell, the team reported that only the cis isomer is pathological, and that Pin1 protects against AD by converting phospho-tau proteins from cis to trans. While some say the findings suggest AD immunotherapy targeting specifically cis phospho-tau is worth a try, others caution that tau’s role is far more complex, with a slew of other molecular alterations potentially influencing the pathological process.

Pin1 binds and isomerizes tau that is phosphorylated at threonine 231 (pT231), one of the earliest phospho-epitopes detected before neurofibrillary tangles form in AD (Luna-Muñoz et al., 2007). Pin1 levels drop in the brains of people with AD (Lu et al., 1999) or frontotemporal dementia (Thorpe et al., 2004). And in mice, lack of Pin1 induces tau and Aβ pathology in otherwise normal animals (ARF related news story on AbstractLiou et al., 2003; ARF related news story on Pastorino et al., 2006), whereas Pin1 overexpression prevents tauopathy in tau transgenic mice (Lim et al., 2008). In human genetic studies, Pin1 polymorphisms that reduce the protein’s expression correlate with higher AD risk (Segat et al., 2007), while gene variants that drive up Pin1 levels delay disease onset (Ma et al., 2010; see Pin1 on AlzGene). “All this suggests that Pin1 protects against AD,” said Lu in an interview with Alzforum. However, without a means to distinguish cis- or trans-specific forms of phosphorylated tau, researchers could not directly show that isomerization influences what tau does, or even what occurs in vivo.

Generating isomer-specific antibodies to cis and trans pT231-tau was the first order of business in the current study. The task was tricky; with only 9 percent of the synthetic peptide in cis form, pT231-tau would work poorly as an immunogen for making cis-specific antibodies. First author Kazuhiro Nakamura and colleagues coaxed the peptide into cis configuration by adding a methylene group that converted proline’s pentagonal structure into a six-member ring. The scientists immunized rabbits with the modified peptide, then collected isomer-specific antibodies by purifying the resulting sera with either cis- or trans-locked phospho-tau peptides. The antibodies showed little cross-reactivity with the opposing isomer in protein binding assays and Western blots. They also immunostained neurons from mouse and Alzheimer’s brains.

While neither conformation of pT231-tau was detected in the brains of healthy elderly, the cis form cropped up in degenerating neurons of people with mild cognitive impairment, and accumulated in dystrophic neurites as disease progresses. In contrast, trans phospho-tau levels were lower, increased only slightly from MCI to AD, and hardly appeared in dystrophic neurites. “This suggested to us that the cis form is pathological and trans is not,” Lu said.

The isomers functioned differently as well. Trans pT231-tau bound tubulin and promoted microtubule assembly in vitro, as normal tau does; however, cis pT231-tau did not. In neuronal cells as well as brain tissue from transgenic mice and MCI patients, cis pT231-tau resisted dephosphorylation and degradation, and aggregated readily, unlike the trans isomer.

Given that Pin1 levels go down in AD, if the cis form of pT231-tau is indeed pathological, then it stands to reason that Pin1 protects against tau pathology by converting phospho-tau from cis to trans, Lu said. This scenario seemed to play out in vivo. When tauopathy mice were crossed to Pin1 transgenic mice (Lim et al., 2008), the double transgenic progeny had more trans pT231-tau and less of the cis form in their brains, compared to tau transgenic littermates with normal Pin1 levels. Conversely, crossing to Pin1 knockouts made tauopathy mice rack up more cis pT231-tau and less of the trans isomer.

“The finding that cis pT231-tau is toxic and that Pin1 can convert it to the non-toxic trans pT231-tau is exciting and has potential to be developed for early diagnosis and perhaps treatment of AD,” suggested Rakez Kayed of the University of Texas Medical Branch, Galveston, in an e-mail to the Alzforum. Julian Thorpe and Stuart Rulten of the University of Sussex, U.K., agreed, saying the data “make a good case for using targeted antibodies against the cis form of phospho-tau in immunotherapy to prevent AD progression.” (See full comment below.)

These scientists and others praised the generation of isomer-specific antibodies as novel tools with broad application. The study “is quite elegant,” commented Charles Glabe of the University of California, Irvine. “It is another good example of how conformation-dependent antibodies give important insights into diseases of protein conformation.” Jürgen Götz of the University of Sydney, Australia, noted, “It will be interesting to see whether the findings for a role of the cis phospho-Thr231 epitope in pathogenesis can be extended to tauopathies other than AD (see full comment below).” Lu said his team has not yet looked for cis and trans phospho-tau in other tauopathies.

However, they have unpublished data showing that the antibodies pick up cis phospho-tau in cerebrospinal fluid of AD patients—and not in normal elderly controls. “We may be able to use this pathologic conformation in CSF as a marker to select high-risk patients for cis-phospho-tau immunotherapy,” Lu said.

Other work by Lu and colleagues suggests Pin1 may also guard against amyloid-β accumulation. Earlier, they found that isomerization of a proline in amyloid precursor protein (APP) curbs Aβ production (Pastorino et al., 2006). In research published this month, the scientists link the Aβ changes with Pin1. One study, which appeared in the March 2 Journal of Biological Chemistry, suggests that the isomerase enhances APP turnover by inhibiting glycogen synthase kinase-3β activity, thereby reducing Aβ production (Ma et al., 2012). Their second paper—posted online March 19 in the Journal of Alzheimer’s Disease—shows that loss of Pin1 function causes APP to move from the cell surface into intracellular compartments to undergo amyloidogenic processing (Pastorino et al., 2012). And in another study—also published this month in the Journal of Alzheimer’s Disease (Giustiniani et al., 2012)—Etienne-Emile Baulieu of INSERM, Paris, France, and colleagues find reduced levels of a different tau-binding prolyl isomerase, FKBP52, in brains of people with AD or frontotemporal dementia with parkinsonism-17 (FTDP-17).—Esther Landhuis

Comments

Comments on News and Primary Papers

The development of cis- and trans-specific anti-tau antibodies by these authors has added very significant tools to the armory of research into AD (and the FTLD tauopathies). They have used these well to demonstrate that, while the trans isoform of tau promotes microtubule assembly, the phosphorylated cis form accumulates in the frontal cortex and hippocampus of patients with MCI and AD. The authors discuss the beneficial effects of cis->trans isomerization catalyzed by Pin1. These demonstrated accumulations of cis-p-tau are as expected, perhaps, but have never been demonstrated before, to our knowledge. We say "expected" because others’ work has shown that Pin1 function is compromised in MCI hippocampus, with the authors concluding that the oxidative inactivation of Pin1 could be involved in the progression from MCI to AD (Butterfield et al., 2006); thus, if Pin1 is the prime mediator of trans-specific tau dephosphorylation, increases in the cis form of tau would be expected in Pin1-deficient MCI or AD brain regions. Also, our own previous work has relevance here, as we showed apparent deficits of Pin1 in FTD brain and also aging-related deficits of the protein in normal brain (Thorpe et al., 2004; Hashemzadeh-Bonehi et al., 2006). This creates a good case for using targeted antibodies against the cis form of p-tau in immunotherapy to prevent AD progression. A caveat to this is that proline isomerization occurs spontaneously in solution from cis to trans and vice versa, and prolyl isomerases such as Pin1 accelerate this isomerization to equilibrium. A drug or antibody that targets and removes cis-p-tau from the environment may thus result in depletion of trans-tau as well as cis-p-tau. Thus, given the beneficial role of trans-tau in microtubule assembly, there may be adverse side effects to this kind of immunotherapy.

The cis/trans configuration of tau adds another layer of complexity to the changes tau undergoes in diseases such as AD, but first of all, this study underlines that it is the serine/threonine-specific hyperphosphorylation of tau rather than any other modification (such as truncation, glycation, or nitration) that causes, at a very early stage, a gain of toxic function of tau and a loss of physiological functions. It will be interesting to see whether the findings for a role of the cis phospho-Thr231 epitope in pathogenesis can be extended to tauopathies other than AD. Furthermore, it will be crucial to determine (as the pThr231-proline motif seems to be the only tau epitope recognized by the isomerase Pin1) whether other phospho-epitopes of tau are also predominantly in the cis configuration in AD, and which enzymes regulate their cis/trans isomerization. Overall, this is an exciting study with interesting antibody tools to be exploited in a wider context.

This intriguing study claims that a localized conformational switch, in the form of the cis/trans conformation of the prolyl bond separating the phosphorylated Threonine 231 (pThr231) and Proline 232 (Pro232) of Tau, carries toxic properties. In order to make this claim, the authors present several polyclonal antibodies that specifically recognize cis or trans forms of this prolyl bond in tau.

In a tau peptide phosphorylated at the Thr231 site, the authors find the prolyl bond equilibrium yields 91 percent in the trans conformation, and 9 percent in cis. Earlier studies (Daly et al., Biochemistry 2000; Smet et al., Febs Lett. 2005) mentioned even lesser amounts of the cis conformer form in slightly longer peptides. Our NMR results on full-length tau point in the same direction, with less than 5 percent of the cis form for this particular prolyl bond (I. Landrieu and GL, unpublished results). An, at first, amazing finding is that the cis antibody in an Elisa test recognizes the natural (majorly trans) pThr-Pro as strongly as the pThr-dimethyl-proline containing one, although the presence of two methyl groups on the proline ring forces the prolyl bond to adopt almost exclusively the cis conformation. The authors’ argument that “cis and trans isomers in the phosphopeptide can be interchanged relatively easily” implies that the cis-specific antibody would basically displace the equilibrium towards the all-cis form by continuously sequestering the soluble cis-form. The same argument can evidently be applied to full-length phospho-Tau, thereby rendering a quantification of the initial cis- content at least hazardous. Equally intriguing is the overlap of the described cis-specific polyclonal antibody epitope with the epitope of the AT180/TG3 antibodies. AT180 was raised against purified human paired helical fragments (Goedert et al., 1994), and would thus be expected to recognize the cis isoform if that is the dominant form in PHFs, as the present study suggests. However, we showed earlier that AT180 recognizes, with nanomolar affinity, tau phosphorylated by CDK2/CycA3 (Amniai et al., BBRC 2011) at the pThr231-Pro232 epitope, where it is almost exclusively in the trans form. An experiment whereby the cis peptide (with the dimethyl-proline) would be probed with the AT180 antibody may bring clarity to this apparent contradiction (M. Mercken, personal communication).

The authors use the modified peptides to confirm the specificity of protein phosphatase 2A (PP2A) for the trans conformation of pThr231-Pro232. Whereas we agree on the trans specificity of PP2A, we did unambiguously show by NMR spectroscopy that PP2A containing the B55 regulatory subunit (which is the one the authors used - see Zhou et al., Mol Cell 2000) does not dephosphorylate the pThr231 position (Landrieu et al., Plos One 2011) but rather the pSer205. Phosphorylation of the Thr231 thereby interferes with the catalytic efficiency of the phosphatase. Because dephosphorylation by PP2A was B55 dependent, we assume that the anchoring of the phospho-Tau substrate to the phosphatase via the regulatory B55 unit is regulated by the phospho-status of Thr231. Pin1 has an effect on the PP2A catalyzed dephosphorylation of phospho-Tau, as it seems to counter-act the negative effect of phospho-Thr231, and thereby stimulates the dephosphorylation of the pSer205 despite the presence of a phospho-Thr231. Whereas it is not clear what exact phosphorylation pattern is generated by the cdc2/CycB kinase used by the authors, disruption of the microtubule (MT) assembly capacity of tau (but not binding to MTs) requires at least three phosphates in the pS202/pS205/pT231/pS235 cluster (Amniai et al., Faseb J 2009). If Pin1 stimulates the dephosphorylation of pSer205, and thereby reduces the phospho content, tau, while still containing the phospho-Thr231, might indeed regain its MT assembly capacity after PP2A treatment (Figure 3G).

In conclusion, the role of Pin1 in changing the phospho-tau conformation remains of considerable interest, but more work is required to evaluate the importance of its isomerase activity on the cis form of the pT231-P232.